CN113025588A - Alcohol dehydrogenase KpADH mutant capable of catalyzing synthesis of piperidine compounds - Google Patents

Alcohol dehydrogenase KpADH mutant capable of catalyzing synthesis of piperidine compounds Download PDF

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CN113025588A
CN113025588A CN202110340244.4A CN202110340244A CN113025588A CN 113025588 A CN113025588 A CN 113025588A CN 202110340244 A CN202110340244 A CN 202110340244A CN 113025588 A CN113025588 A CN 113025588A
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文雨
华希锐
干淼钰
徐芬
钱贵猛
倪晔
周婕妤
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Jiangnan University
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Abstract

The invention discloses an alcohol dehydrogenase KPADH mutant capable of catalyzing and synthesizing piperidine compounds, and belongs to the technical field of genetic engineering and enzyme engineering. The KPADH mutant is mutated on the basis of a recombinant alcohol dehydrogenase KPADH derived from Kluyveromyces polyspora, and the constructed alcohol dehydrogenase KPADH mutant has remarkable improvement on the performance of catalyzing and oxidizing pyrrolidine, piperidine and alcohol, can ensure that the conversion rate reaches 99.9 percent within 6 hours when rac-NBHP is taken as a substrate, can convert and produce NBPO by taking high-concentration rac-NBHP as a substrate, and has higher industrial application value compared with the wild catalytic performance, particularly the application in preparing medicament ibrutinib.

Description

Alcohol dehydrogenase KpADH mutant capable of catalyzing synthesis of piperidine compounds
Technical Field
The invention relates to an alcohol dehydrogenase KPADH mutant capable of catalyzing and synthesizing piperidine compounds, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Ibrutinib (irutinib, formerly known as Imbruvica) is a new anti-lymphoma drug of Bruton's Tyrosine Kinase (BTK) inhibitor. Compared with other medicines, the ibrutinib has the characteristics of high efficiency, specificity, irreversibility and high targeting property on BTK. The precursor N-acryloyl piperidine ring with optical activity has complex chemical synthesis steps, high cost, low yield and the like. Therefore, starting from a low-cost substrate 3-hydroxypyridine, the chemical synthesis is combined with biological enzyme catalysis, the synthesis bottleneck of a precursor compound N-Boc-3-piperidone is firstly broken through, and then the de novo synthesis of (S) -N-Boc-3-hydroxypiperidine ((S) -NBHP) is realized through coupling KPADH catalyzed asymmetric reduction reaction. The chemical biological catalysis method has the advantages of greatly reduced cost, high substrate specificity, mild reaction conditions, environmental friendliness and the like, and has research and application values.
Alcohol dehydrogenase KPADH (Kluveromyces polyspora) belongs to the Alcohol Dehydrogenase (ADH) and aldone Reductase superfamily (AKR), which can use coenzyme NAD (P)+And a reducing agent (usually glucose, formate or isopropanol) into the chiral molecule. It is widely found in animal and plant, bacteria and other tissues in nature. Alcohol dehydrogenases can be divided into Short-chain Dehydrogenase Superfamily (SDR) independent of metal ions and Zn-dependent on metal ions according to the length of peptide chain and dependence on metal2+Medium-chain Dehydrogenase superfamily (M-chain Dehydrogenase/Reductase)DR). In recent years, there have been many reports on the preparation of chiral benzyl alcohol by alcohol dehydrogenase. KPADAH is a known enzyme with the capability of oxidizing a large-potential hindered alcohol substrate, has wide application in industry, and provides wide application prospect for future industrial biocatalysis.
Although the KPAADH has great application and research value, the activity of the specific catalytic NBHP of the KPAADH screened from wild bacteria is low, so that the enzyme cannot be further applied to industrial production, and the development and application are greatly reduced.
Disclosure of Invention
In view of the problems that the activity of the wild KPADH is low and the requirement of industrial production cannot be met, the invention carries out heterologous expression and site-specific mutagenesis modification on the recombinant alcohol dehydrogenase KPADH from Kluyveromyces Kluyveromyces polyspora, improves the capability of the modified KPADH to oxidize NBHP, and has important significance for industrial application and popularization of the KPADH.
The invention provides an alcohol dehydrogenase mutant, which takes alcohol dehydrogenase with an amino acid sequence shown as SEQ ID NO.1 as a parent and mutates one or more sites of 128 th site, 131 th site, 161 th site, 165 th site and 196 th site of the parent.
In one embodiment, the alanine at position 128 is mutated to cysteine or valine, the phenylalanine at position 161 is mutated to tryptophan, and the serine at position 196 is mutated to glycine.
In one embodiment, the mutant is capable of specifically catalyzing the synthesis of N-Boc-3-piperidone (NBPO) from NBHP, and then realizing a KPADH mutant synthesized de novo from the drug ibrutinib intermediate (S) -NBHP.
In one embodiment, the mutant is a mutation of alanine at position 128 to cysteine based on the amino acid sequence of SEQ ID NO:1, designated M1.
In one embodiment, the mutant is a mutation of alanine at position 128 to valine based on the amino acid sequence of SEQ ID NO:1, designated M2.
In one embodiment, the mutant is obtained by mutating phenylalanine at position 161 to tryptophan on the basis of the amino acid sequence of seq id No.1, and is designated as M3.
In one embodiment, the mutant is obtained by mutating serine at position 196 to glycine, designated as M4, based on the amino acid sequence of seq id No. 1.
In one embodiment, the mutant is obtained by mutating alanine at position 128 to cysteine, and simultaneously mutating serine at position 196 to glycine on the basis of the amino acid sequence of SEQ ID NO.1, and is named M5.
In one embodiment, the mutant is obtained by mutating alanine at position 128 to valine on the basis of the amino acid sequence of SEQ ID NO:1, and simultaneously mutating phenylalanine at position 161 to serine, which is designated M6.
In one embodiment, the mutant is obtained by mutating serine at position 196 to glycine and alanine at position 128 to valine on the basis of the amino acid sequence of SEQ ID NO.1 and is designated M7.
In one embodiment, the mutant is obtained by mutating phenylalanine at position 161 to tryptophan and simultaneously mutating alanine at position 128 to cysteine on the basis of the amino acid sequence of SEQ ID NO.1 and is named M8.
The present invention provides a gene encoding the alcohol dehydrogenase mutant.
The invention provides a recombinant plasmid carrying the gene.
In one embodiment, the recombinant plasmid uses a pET series vector as a starting vector.
The invention provides microbial cells expressing the mutants, or carrying the recombinant plasmids.
In one embodiment, the microbial cell is a host escherichia coli.
The invention provides a method for producing piperidone compounds, which takes the piperidone compounds as a substrate and the mutant as a catalyst.
In one embodiment, the concentration of the piperidine compound is 10 to 60 mM.
In one embodiment, the piperidines include rac-N-tert-butoxycarbonyl-3-hydroxypiperidine, N-tert-butoxycarbonyl-4-hydroxypiperidine, 3-hydroxypiperidine.
In one embodiment, the reaction is carried out at pH 7.0-8.0.
The invention provides the application of the mutant, the gene, the recombinant plasmid or the microbial cell in the production of chiral alcohol compounds.
The invention provides the application of the mutant, the gene, the recombinant plasmid or the microbial cell in the production of ketone compounds.
In one embodiment, the ketone compound is converted using racemic alcohol as a substrate.
In one embodiment, the ketone compound comprises a piperidone compound.
The invention has the following beneficial effects:
the KPADH mutant is mutated on the basis of a recombinant alcohol dehydrogenase KPADH derived from Kluyveromyces polyspora, and the constructed alcohol dehydrogenase KPADH mutant has remarkable improvement on the performance of catalyzing and oxidizing pyrrolidine, piperidine and alcohol, can ensure that the conversion rate reaches 99.9 percent within 6 hours when rac-NBHP is taken as a substrate, can convert and produce NBPO by taking high-concentration rac-NBHP as a substrate, and has higher industrial application value compared with the wild catalytic performance, particularly the application in preparing medicament ibrutinib.
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FIG. 1 shows the electrophoresis of the nucleic acids of the whole plasmid PCR of wild-type and alcohol dehydrogenase mutants M1-M7.
Detailed Description
The alcohol dehydrogenase enzyme activity detection method comprises the following steps:
the activity measuring principle is as follows: according to the characteristic absorption peak of NAD (P) H at 340nm, alcohol dehydrogenase generates or consumes NAD (P) H during the oxidation or reduction reaction. Thus, enzyme activity can be calculated indirectly using changes in NAD (P) H at 340 nm.
The total reaction system was 200 μ L, including: 20mM NAD (P)+100mM substrate rac-NBHP, sodium phosphate buffer (PBS, l00 mM, pH 7.0), mixing well, incubating at 30 deg.C for 2min, adding appropriate amount of enzyme solution, and detecting the change of light absorption value at 340 nm.
The enzyme activity calculation formula is as follows: u is EW multiplied by V multiplied by 103/(6200×L)
In the formula, EW: change value of absorbance at 340nm within 1 min; v: total volume of reaction solution (mL); 6220: molar extinction coefficient (L. mol)-1·cm-1) (ii) a L: optical path length (cm).
The enzyme activity is defined as follows:
one enzyme activity unit (1U) is defined as catalyzing 1. mu. mol NAD (P) per minute under the above-mentioned activity measuring conditions+The amount of enzyme required to produce NAD (P) H.
Example 1: construction of alcohol dehydrogenase mutant Gene and recombinant expression transformant
And (3) carrying out site-directed mutagenesis on the amino acid residues at 128, 131, 161 and 196 by adopting a whole plasmid PCR method to construct an iterative combinatorial mutant. Connecting a coding gene of alcohol dehydrogenase KPADH with a nucleotide sequence shown as SEQ ID NO.2 to a polyclonal enzyme cutting site of a pET28a vector; based on the sequence of alcohol dehydrogenase KPADH (amino acid sequence shown in SEQ ID NO. 1), the primers were designed as follows (all described in 5 '-3' direction, underlined indicates mutation site):
m1 (using pET28a-KPADH recombinant plasmid as template):
A128C-F:ACTGCTTCTTATTGTTCAATT，
A128C-R:GGTCATAATTGAACAATAAGA；
m2 (using pET28a-KPADH recombinant plasmid as template):
A128V-F:ACTGCTTCTTATGTTTCAATT，
A128V-R:GGTCATAATTGAAACATAAGA；
m3 (using pET28a-KPADH recombinant plasmid as template):
F161W-F:TATGAAAATGTCTGGACTGCT，
F161W-R:ACAATAAGCAGTCCAGACATT；
m4 (using pET28a-KPADH recombinant plasmid as template):
S196G-F:ACTATCCACCCAGGTTTCGTT，
S196G-R:TCCGAAAACGAAACCTGGGTG；
m5 (using M1 recombinant plasmid as template):
S196G-F:ACTATCCACCCAGGTTTCGTT，
S196G-R:TCCGAAAACGAAACCTGGGTG；
m6 (using M2 recombinant plasmid as template):
F161V-F:TATGAAAATGTCGTTACTGCT，
F161V-R:ACAATAAGCAGTAACGACATT；
m7 (using M3 recombinant plasmid as template):
A128V-F:ACTGCTTCTTATGTTTCAATT，
A128V-R:GGTCATAATTGAAACATAAGA；
m8 (using M4 recombinant plasmid as template):
A128C-F:ACTGCTTCTTATTGTTCAATT，
A128C-R:GGTCATAATTGAACAATAAGA。
the PCR reaction system is as follows: the PCR reaction system (20. mu.L) contained 0.4. mu.L of KOD enzyme (2.5U/mL), 0.3. mu.L of template (5-20ng), 2. mu.L of dNTP, 2. mu.L of 10 × reactionbuffer, 0.3. mu.L of each of the upstream and downstream primers, and ddH2Make up to 20. mu.L of O.
The PCR amplification procedure was: pre-denaturation at 95 deg.C for 6min, denaturation at 98 deg.C for 10s, annealing at 50 deg.C for 30s, extension at 68 deg.C for 3.5min, circulation for 20 times, extension at 68 deg.C for 10min, and storage at 4 deg.C. After confirming the successful amplification by 1% agarose nucleic acid gel electrophoresis, 10. mu.L of the digested PCR reaction solution was transferred to 100. mu.L of E.coli BL21(DE3) competent cells by the heat transfer method using Quick Cut Dpn I enzyme incubated at 37 ℃ for 1 hour, and uniformly spread on an LB solid plate containing 50. mu.g/mL kanamycin, and incubated at 37 ℃ for 12 hours. FIG. 1 shows the electrophoresis of the whole plasmid PCR nucleic acid of wild-type and alcohol dehydrogenase mutants M1-M7, and it can be seen that the PCR product is between 10000bp and 3000bp (the band size is 5000 bp).
Example 2: expression and purification of alcohol dehydrogenase and its mutant
The deposited WT KPADH and the mutation in example 2 were addedThe glycerol strain of the variant was inoculated into LB medium containing 50. mu.g/mL of kanamycin, shake-cultured at 37 ℃ for 8 hours at 120rpm, and the activated seed solution was transferred to 30mL of LB medium containing 50. mu.g/mL of kanamycin in an inoculum size of 1% (v/v). When OD is reached600When the concentration reaches 0.8, 0.2mM IPTG is added for induction, the induction temperature is 25 ℃, after 8 hours of induction, the thalli of the high-efficiency expression recombinant alcohol dehydrogenase mutant are obtained by centrifugation at 8000rpm for 10 minutes, the collected thalli are suspended in potassium phosphate buffer (100mM, pH 6.0) and are crushed by ultrasound.
The cell lysate was centrifuged at 8000rpm for 20min at 4 ℃.
The column used for purification was a nickel affinity column, HisTrap FF crude, and affinity chromatography was performed using a histidine tag on the recombinant protein. Gradient elution step: (1) sample treatment: collecting cell disruption solution after ultrasonic centrifugation, and filtering with 0.22 μm membrane to remove impurities; (2) and (3) nickel column treatment: the 20% ethanol used for column storage was eluted naturally, washed with 10mL of ultrapure water, followed by 10mL of suction-filtered PBS buffer (100mM, pH 7.0) to equilibrate the column; (3) loading: slowly pouring the pretreated sample into a chromatographic column, collecting effluent liquid, and passing through the column again; (4) and (3) elution: the enzyme activity was measured by sequentially eluting with PBS buffers (100mM, pH 7.0) containing different imidazole concentrations (20mM, 50mM, 100mM, 150mM, 300mM), eluting 5mL each, and collecting the permeates at the time of elution with different imidazole concentrations. The KPAADH and the mutant thereof can be completely eluted when the concentration of imidazole reaches 300mM in the elution process. Samples eluted at 300mM imidazole were collected and analyzed by SDS-PAGE for bands of interest.
Example 3: substrate spectrum activity analysis of alcohol dehydrogenase mutant
The alcohol dehydrogenase mutants obtained in example 2 were used as catalysts, and the potential chiral hydroxy compounds were used as substrates to demonstrate the ability of each mutant to oxidize the potential chiral hydroxy compounds: examples of potential chiral hydroxy compounds examined include N-t-butoxycarbonyl-3-hydroxypyrrolidine (N-Boc-3-hydroxypyrolidine), rac-N-t-butoxycarbonyl-3-hydroxypiperidine (rac-N-Boc-3-hydroxypiperidine-dine, rac-NBHP), N-t-butoxycarbonyl-4-hydroxypiperidine (N-Boc-4-hydroxypiperidine), 1-Phenyl-ethanol (1-Phenyl-ethanol), 4-chlorophenylethanol (4' -chlorophenylethyl-ethanol), (4-chlorophenyl) -pyridin-2-yl-methanol ((4-Chloro-Phenyl) -pyridine-2-y 1-methanol), 3-hydroxypiperidine (3-Pipe-pyridine).
TABLE 1 substrate Profile Activity of alcohol dehydrogenase mutants
Figure BDA0002999291880000051
Figure BDA0002999291880000061
As can be seen from Table 1, M8 showed the highest activity for rac-N-t-butyloxycarbonyl-3-hydroxypiperidine (rac-NBHP) compared to WT.
Example 4: preparation of NBPO by oxidizing rac-NBHP with alcohol dehydrogenase mutant
10mL of biocatalytic system: 100mM potassium phosphate buffer (pH 7.0), the alcohol dehydrogenase mutant M8 obtained in example 2 and the wild type KPADH 10g/L, rac-NBHP 10mM, 20mM and 50mM were added. The reaction was carried out at 30 ℃ and 200rpm, with a constant pH of 7.5.
The results of the conversion analysis during the reaction are shown in tables 2 and 3, and the wild-type dehydrogenase was able to achieve a higher conversion at a lower substrate concentration, but had a limited conversion ability. When the concentration of rac-NBHP is 20mM, the wild-type KPADH and the mutant M8 only need 6h to achieve 99.9% conversion, while the wild-type requires 12h and 24h to react for 12h to achieve nearly 99.9% conversion.
TABLE 2 wild-type alcohol dehydrogenase KpAADH Oxidation rac-NBHP
Figure BDA0002999291880000062
TABLE 3 alcohol dehydrogenase mutant M8 Oxidation of rac-NBHP
Figure BDA0002999291880000063
Figure BDA0002999291880000071
From this, it can be seen that the alcohol dehydrogenase mutant enzyme M8 has very good performance in the conversion of rac-NBHP.
Comparative example 1
The 127 th mutation of the alcohol dehydrogenase with the amino acid sequence shown as SEQ ID NO.1 is leucine, the 128 th mutation is threonine, the 131 th mutation is histidine, and the 165 th mutation is tyrosine, phenylalanine and proline, and the result shows that the catalytic specific activity to the substrate is less than that of the wild bacteria.
The results of mutating the 131 th site of the alcohol dehydrogenase with the amino acid sequence shown as SEQ ID NO.1 into histidine, the 161 th site into tryptophan and the 165 th site into serine show that the catalytic specific activity to a substrate is not improved much.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> alcohol dehydrogenase KPADH mutant capable of catalyzing synthesis of piperidine compounds
<130> BAA210207A
<160> 2
<170> PatentIn version 3.3
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<211> 334
<212> PRT
<213> Kluyveromyces polyspora
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Asp Ile Phe Lys Lys His Gly Lys Glu Ile Lys Tyr Val Ile His Ala
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Lys Phe Val Lys Glu Asn Ser Asp Ala Val Lys Phe Lys Leu Thr Thr
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Ile His Pro Ser Phe Val Phe Gly Pro Gln Asn Phe Asp Glu Asp Val
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Phe Thr Pro Phe His Lys Thr Ile His Asp Thr Val Tyr Gln
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<210> 2
<211> 1002
<212> DNA
<213> Kluyveromyces polyspora
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ttgaaacaat ataataatcc taatttgtct tatgaaattg tacctgaaat agcaaactta 180
gatgcttttg atgacatttt taagaaacat ggtaaggaaa taaaatatgt cattcatgca 240
gcttcaccag tgaacttcgg cgcaaaagat ttggaaaaag atttagttat tcctgccatt 300
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tttactcctt tccataaaac aattcatgac actgtctatc aa 1002

Claims (10)

1. An alcohol dehydrogenase mutant characterized in that the alcohol dehydrogenase mutant takes an alcohol dehydrogenase of which the amino acid sequence is shown as SEQ ID NO.1 as a parent and mutates one or more of the 128 th site, 131 th site, 161 th site, 165 th site and 196 th site of the parent.
2. The alcohol dehydrogenase mutant according to claim 1, wherein alanine at position 128 is mutated to cysteine or valine, phenylalanine at position 161 is mutated to serine, and serine at position 196 is mutated to glycine.
3. A gene encoding the alcohol dehydrogenase mutant of claim 1 or 2.
4. A recombinant plasmid carrying the gene of claim 3.
5. A microbial cell expressing a mutant according to claim 1 or 2, or carrying a recombinant plasmid according to claim 4.
6. A method for producing a piperidone compound, which comprises using a piperidine compound as a substrate and the mutant of claim 1 or 2 as a catalyst.
7. The method of claim 6, wherein the concentration of the piperidine compound is 10-60 mM.
8. The method of claim 6, wherein the piperidine compound comprises rac-N-tert-butoxycarbonyl-3-hydroxypiperidine, N-tert-butoxycarbonyl-4-hydroxypiperidine, 3-hydroxypiperidine.
9. The method according to claim 8, wherein the reaction is carried out at pH7.0 to 8.0.
10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant plasmid according to claim 4 or 5, or the microbial cell according to claim 6 for the production of chiral alcohol compounds and piperidones.
CN202110340244.4A 2021-03-30 2021-03-30 Alcohol dehydrogenase KpADH mutant capable of catalyzing synthesis of piperidine compounds Pending CN113025588A (en)

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CN108504641A (en) * 2018-02-12 2018-09-07 江南大学 Alcohol dehydrogenase mutant and its application in the synthesis of double aryl chiral alcohols
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Application publication date: 20210625